Journal of Investigative Surgery, Volume 3. pp. 141-145 Printed in the UK. AU rights reserved.
0894-1939i9053.00 t .OO Copyright 0 1990 Taylor & Francis
Methodologies, Devices, and New Surgical Concepts
Warfarin Sodium for Anticoagulation of Atherosclerotic Miniature Swine
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
DOUGLAS M. CROMEENS, DVM The University of Texas System Cancer Center at M.D. Anderson Hospital Houston, TX 77030
GEORGE P. RODGERS, MD STEVEN T. MINOR, MD Baylor College of Medicine Houston, TX 77030 Abstract Warfarin sodium (Coumadin) has been used as an effective anticoagulating agent in human medicine for many years, although careful monitoring of its effects are necessary to avoid excessive anticoagulation. Previous experience with this drug for chronic anticoagulation therapy in miniature swine has been limited. The effect of warfarin sodium was studied by measuring prothrombin time in twelve 8-month-old Hanford miniature swine. The pigs had been fed a high-cholesterol diet and had undergone a prior coronary artery abrasion procedure for development of an atherosclerotic coronary disease model. Atherosclerosis was induced by feeding a high-cholesterol diet. Baseline prothrombin time ranged from 12.8 t o 15.0 s (13.7 s mean). Prothrombin time was determined daily for the first 5 days of treatment and at least twice weekly thereafter until the animals were sacrificed. Animals received warfarin for 37-41 days. Prothrombin time could be increased 33-50% by once daily oral administration of warfarin 0.04-0.08 mglkg. Oral administration of more than 0.08 mglkg as a maintenance dose resulted in the death of two pigs. Most animals responded well to 0.08 mglkg for the first 3 days of treatment followed by a maintenance dose of 0.06 mglkg. Dosage was adjusted periodically when prothrombin times exceeded 50% above baseline. It is our experience that monitoring prothrombin time at least twice weekly and adjusting the maintenance dose can eliminate death losses due to warfarin intoxication.
Keywords: Warfarin, Coumadin, anticoagulation, miniature swine, atherosclerosis.
Warfarin sodium has been used in human medicine for many years. However, the experience with warfarin in veterinary medicine is limited and, more specifically, Address correspondence to Douglas M. Cromeens, DVM, Department of Veterinary Medicine and Surgery, The University of Texas System, Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. 141
D.M. Cromeens et al.
142
safe and effective dosage regimes for its use in miniature swine have not been established. This study describes the development of an effective technique for both the administration of warfarin sodium and monitoring of coagulation status in atherosclerotic miniature swine.
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
Materials and Methods Animals used in this study consisted of fourteen 8-month-old male Hanford minipigs. These animals were obtained from the University of Texas System Cancer Center, Division of Veterinary Resources' established herd. Twelve pigs had been fed and were maintained on a 2% cholesterol, 15% total fat, and 1.5% sodium cholate diet for 4 months. Total serum cholesterol ranged from 472 to 962 mg% (684 mg% mean) before and during warfarin therapy. The animals averaged 23 kg at the time warfarin therapy was instituted. Pigs were individually housed in conventional facilities and received humane care in compliance with The Guide for the Care and Use of Laboratory Animals.' All pigs in the study had 18-gauge silastic catheters placed in the anterior vena cava via the external jugular vein. The catheter was exteriorized in the dorsal cervical area just caudal to the pinna of the ear. Catheters were flushed twice daily with 20 mL of heparinized saline (4 U/mL) after administration of 5 mg/kg ampicillin through the catheter. All blood samples used for monitoring prothrombin time (PT) were drawn from the conscious animal through this catheter. PT determinations were performed on sodium citrate blood samples using a one-stage prothrombin time technique3 and thromboplastin C provided by Baxter Dade. PT was monitored daily for the first 4-6 days of therapy and then reduced to two times weekly until the pigs were taken out of the study. Oral warfarin sodium (Coumadin, 2-mg tabs), 0.08 mg/kg, was administered orally once daily PIG #40 3mg I
1
--
omg 20
-
10
-
n
n 8
2 4 6 8 1 0
20
Days
30
40
on Warfarin Sodium
Figure 1. Typical prothrombin time response of an 8-month-old 25-kg minipig to 0.04-0.08 mdkg per day oral sodium warfarin.
Anticoagulation of Minipigs
143
for the first 2-4 days. When the PT increased 30-50% above baseline, the dosage was reduced to 0.04-0.08 mg/kg daily. The dosage of warfarin was adjusted as necessary to maintain the extended PT in the stated range, with the dose never exceeding 0.08 mg/kg per day. Administration of warfarin was discontinued temporarily when the PT exceeded 150% of the baseline figure.
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
Results Ten pigs were treated with warfarin for 37-41 days. Two pigs died at 9 days from complications of surgical manipulations unrelated to the warfarin therapy. All animals tolerated the medication well with no sign of warfarin intoxication. Prothrombin times were held 30-50% above normal by adjusting the warfarin dose between 0.04 and 0.08 mg/kg per day (Fig 1). Two pigs that were used in a pilot study to establish beginning doses of warfarin were given 0.10-0.15 mg/kg per day and died after 10 days of therapy from warfarin intoxication. Symptoms before death included bilateral epistaxis, melena, and subcutaneous hematomas. Postmortem findings revealed hemorrhage in the lungs, and one of the pigs had a 3-cm gastric ulcer. Both pigs had prothrombin times of 32 and 25 s immediately before death.
Discussion “Effective therapeutic levels with minimal complications can best be achieved in cooperative and well-instructed patients, who keep the doctor informed of their status between visits.”2 This statement best explains the difficulties that anticoagulating miniature swine can pose. Behavioral characteristics, difficulty in obtaining peripheral blood samples, and a narrow safe dosage range all work together to make successful anticoagulation of miniature swine without high morbidity/mortality from warfarin intoxication a challenge. The recommended initial human dose of warfarin is 10-15 mg daily for 2-3 days and thereafter adjusted to prothrombin time response.2 Our experience in the pilot study and the only available reference that addressed the use of warfarin in pigs4 illustrated the need to begin the animals on a lower initial dose than what would be considered safe in the human. Warfarin 0.08 mg/kg per day was found to be well tolerated and did not produce abrupt increases in PT. The average maintenance dose of 0.06 mg/kg per day compares very closely with the dosage required in humans to prolong the F T 30-50%. Adequate anticoagulation is considered accomplished in the human when the PT is extended 30-50% above baseline (16-18 s ) . This ~ more conservative approach to anticoagulation has been credited with decreased incidence of bleeding complications associated with warfarin therapy.6 It was our experience that the minipigs show severe symptoms of warfarin intoxication with PT values as low as 25 s (2.0 times normal) and would therefore not lend themselves to the more aggressive anticoagulation therapy that has been used in human medicine in the past. Maintaining the PT at 1.3- 1.5 times normal in the pig proved to be safe with no morbidity or mortality. It is recommended that human patients receiving warfarin therapy have their prothrombin time monitored daily until they are stabilized in the therapeutic range (1.5-2.5 times normal).* Due to the pig’s observed sensitivity to warfarin,
D . M . Cromeens et al.
144
PIG #11
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
1
20
-
10
-
8
" " ' 2 4 6 8 1 0
20 30 Days on Warlarln Sodlum
40
Figure 2. Response of a 24-kg minipig demonstrating variation of prothrombin time to a given dose of sodium warfarin.
and they appear to succumb to bleeding complications at lower F T levels than the human, we believe that monitoring the PT daily for the first 4-7 days and twice weekly thereafter is an essential part of successful anticoagulation therapy in the pig. The importance of monitoring throughout therapy is illustrated in Figure 2. It was not uncommon for individual pigs to show great variation in the dose necessary to stay in the therapeutic range without inducing bleeding complications. This is complicated by the difficulty of obtaining adequate samples from the conscious animal. The use of a silastic catheter in the external jugular vein or some other device for daily sampling of peripheral blood without exciting the animal facilitates the monitoring of PT in the conscious pig and is highly recommended.
Conclusion The miniature pig is gaining in popularity in cardiovascular research, and the ease with which atherosclerosis can be induced adds to its usefulness as a model of human coronary artery d i ~ e a s e The . ~ ~ results ~ of this study have shown their added utility in studies that require the administration of warfarin sodium.
References 1. National Institutes of Health. Guide for the Care and Use of Laboratory Animals. NIH
Publication 85-23; 1985. 2. Physicians Desk Reference. Oradell, NJ: Medical Economics; 1989: pp 902-904. 3. Quick AJ. Hemorrhagic diseases and thrombosis. Philadelphia: Lea 8z Febiger; 1966. 4. Davilla JC, Edds GT, Osuna 0. Modification of the effects of aflatoxin BI and warfarin in young pigs given selenium. Am J Vet Res. 1983;44:1877-1883. 5 . Koph GS, Graeme LH, Geha AS: Long-term performance of the St. Jude Medical
Anticoagulation of Minipigs
145
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
valve: Low incidence of thromboembolism and hemorrhagic complications with modest doses of warfarin. Circulation. 1987;76(supplIII):III-132-III-136. 6. Hirsh J. Is the dose of warfarin prescribed by American physicians unnecessarily high? Arch Intern Med. 1987;147:769-771. 7. Kim DN, Lee KT, Schmee J , Thomas WA. Quantification of intimal cell masses and atherosclerotic lesions in coronary arteries of control and hyperlipidemic swine. Atherosclerosis. 1984;52:115- 122. 8. Thomas WA, Kim DN, Lee KT, Reiner JM, Schmee J. Population dynamics of arterial cells during atherogenesis. XIII. Mitogenic and cytotoxic effects of a hyperlipidemic diet on cells in advanced lesions in the abdominal aortas of swine fed HL diet for 270-345 days. Exp Mol Pathol. 1983;39:257-270.
0894-1939i9053.00 t .OO Copyright 0 1990 Taylor & Francis
Methodologies, Devices, and New Surgical Concepts
Warfarin Sodium for Anticoagulation of Atherosclerotic Miniature Swine
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
DOUGLAS M. CROMEENS, DVM The University of Texas System Cancer Center at M.D. Anderson Hospital Houston, TX 77030
GEORGE P. RODGERS, MD STEVEN T. MINOR, MD Baylor College of Medicine Houston, TX 77030 Abstract Warfarin sodium (Coumadin) has been used as an effective anticoagulating agent in human medicine for many years, although careful monitoring of its effects are necessary to avoid excessive anticoagulation. Previous experience with this drug for chronic anticoagulation therapy in miniature swine has been limited. The effect of warfarin sodium was studied by measuring prothrombin time in twelve 8-month-old Hanford miniature swine. The pigs had been fed a high-cholesterol diet and had undergone a prior coronary artery abrasion procedure for development of an atherosclerotic coronary disease model. Atherosclerosis was induced by feeding a high-cholesterol diet. Baseline prothrombin time ranged from 12.8 t o 15.0 s (13.7 s mean). Prothrombin time was determined daily for the first 5 days of treatment and at least twice weekly thereafter until the animals were sacrificed. Animals received warfarin for 37-41 days. Prothrombin time could be increased 33-50% by once daily oral administration of warfarin 0.04-0.08 mglkg. Oral administration of more than 0.08 mglkg as a maintenance dose resulted in the death of two pigs. Most animals responded well to 0.08 mglkg for the first 3 days of treatment followed by a maintenance dose of 0.06 mglkg. Dosage was adjusted periodically when prothrombin times exceeded 50% above baseline. It is our experience that monitoring prothrombin time at least twice weekly and adjusting the maintenance dose can eliminate death losses due to warfarin intoxication.
Keywords: Warfarin, Coumadin, anticoagulation, miniature swine, atherosclerosis.
Warfarin sodium has been used in human medicine for many years. However, the experience with warfarin in veterinary medicine is limited and, more specifically, Address correspondence to Douglas M. Cromeens, DVM, Department of Veterinary Medicine and Surgery, The University of Texas System, Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. 141
D.M. Cromeens et al.
142
safe and effective dosage regimes for its use in miniature swine have not been established. This study describes the development of an effective technique for both the administration of warfarin sodium and monitoring of coagulation status in atherosclerotic miniature swine.
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
Materials and Methods Animals used in this study consisted of fourteen 8-month-old male Hanford minipigs. These animals were obtained from the University of Texas System Cancer Center, Division of Veterinary Resources' established herd. Twelve pigs had been fed and were maintained on a 2% cholesterol, 15% total fat, and 1.5% sodium cholate diet for 4 months. Total serum cholesterol ranged from 472 to 962 mg% (684 mg% mean) before and during warfarin therapy. The animals averaged 23 kg at the time warfarin therapy was instituted. Pigs were individually housed in conventional facilities and received humane care in compliance with The Guide for the Care and Use of Laboratory Animals.' All pigs in the study had 18-gauge silastic catheters placed in the anterior vena cava via the external jugular vein. The catheter was exteriorized in the dorsal cervical area just caudal to the pinna of the ear. Catheters were flushed twice daily with 20 mL of heparinized saline (4 U/mL) after administration of 5 mg/kg ampicillin through the catheter. All blood samples used for monitoring prothrombin time (PT) were drawn from the conscious animal through this catheter. PT determinations were performed on sodium citrate blood samples using a one-stage prothrombin time technique3 and thromboplastin C provided by Baxter Dade. PT was monitored daily for the first 4-6 days of therapy and then reduced to two times weekly until the pigs were taken out of the study. Oral warfarin sodium (Coumadin, 2-mg tabs), 0.08 mg/kg, was administered orally once daily PIG #40 3mg I
1
--
omg 20
-
10
-
n
n 8
2 4 6 8 1 0
20
Days
30
40
on Warfarin Sodium
Figure 1. Typical prothrombin time response of an 8-month-old 25-kg minipig to 0.04-0.08 mdkg per day oral sodium warfarin.
Anticoagulation of Minipigs
143
for the first 2-4 days. When the PT increased 30-50% above baseline, the dosage was reduced to 0.04-0.08 mg/kg daily. The dosage of warfarin was adjusted as necessary to maintain the extended PT in the stated range, with the dose never exceeding 0.08 mg/kg per day. Administration of warfarin was discontinued temporarily when the PT exceeded 150% of the baseline figure.
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
Results Ten pigs were treated with warfarin for 37-41 days. Two pigs died at 9 days from complications of surgical manipulations unrelated to the warfarin therapy. All animals tolerated the medication well with no sign of warfarin intoxication. Prothrombin times were held 30-50% above normal by adjusting the warfarin dose between 0.04 and 0.08 mg/kg per day (Fig 1). Two pigs that were used in a pilot study to establish beginning doses of warfarin were given 0.10-0.15 mg/kg per day and died after 10 days of therapy from warfarin intoxication. Symptoms before death included bilateral epistaxis, melena, and subcutaneous hematomas. Postmortem findings revealed hemorrhage in the lungs, and one of the pigs had a 3-cm gastric ulcer. Both pigs had prothrombin times of 32 and 25 s immediately before death.
Discussion “Effective therapeutic levels with minimal complications can best be achieved in cooperative and well-instructed patients, who keep the doctor informed of their status between visits.”2 This statement best explains the difficulties that anticoagulating miniature swine can pose. Behavioral characteristics, difficulty in obtaining peripheral blood samples, and a narrow safe dosage range all work together to make successful anticoagulation of miniature swine without high morbidity/mortality from warfarin intoxication a challenge. The recommended initial human dose of warfarin is 10-15 mg daily for 2-3 days and thereafter adjusted to prothrombin time response.2 Our experience in the pilot study and the only available reference that addressed the use of warfarin in pigs4 illustrated the need to begin the animals on a lower initial dose than what would be considered safe in the human. Warfarin 0.08 mg/kg per day was found to be well tolerated and did not produce abrupt increases in PT. The average maintenance dose of 0.06 mg/kg per day compares very closely with the dosage required in humans to prolong the F T 30-50%. Adequate anticoagulation is considered accomplished in the human when the PT is extended 30-50% above baseline (16-18 s ) . This ~ more conservative approach to anticoagulation has been credited with decreased incidence of bleeding complications associated with warfarin therapy.6 It was our experience that the minipigs show severe symptoms of warfarin intoxication with PT values as low as 25 s (2.0 times normal) and would therefore not lend themselves to the more aggressive anticoagulation therapy that has been used in human medicine in the past. Maintaining the PT at 1.3- 1.5 times normal in the pig proved to be safe with no morbidity or mortality. It is recommended that human patients receiving warfarin therapy have their prothrombin time monitored daily until they are stabilized in the therapeutic range (1.5-2.5 times normal).* Due to the pig’s observed sensitivity to warfarin,
D . M . Cromeens et al.
144
PIG #11
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
1
20
-
10
-
8
" " ' 2 4 6 8 1 0
20 30 Days on Warlarln Sodlum
40
Figure 2. Response of a 24-kg minipig demonstrating variation of prothrombin time to a given dose of sodium warfarin.
and they appear to succumb to bleeding complications at lower F T levels than the human, we believe that monitoring the PT daily for the first 4-7 days and twice weekly thereafter is an essential part of successful anticoagulation therapy in the pig. The importance of monitoring throughout therapy is illustrated in Figure 2. It was not uncommon for individual pigs to show great variation in the dose necessary to stay in the therapeutic range without inducing bleeding complications. This is complicated by the difficulty of obtaining adequate samples from the conscious animal. The use of a silastic catheter in the external jugular vein or some other device for daily sampling of peripheral blood without exciting the animal facilitates the monitoring of PT in the conscious pig and is highly recommended.
Conclusion The miniature pig is gaining in popularity in cardiovascular research, and the ease with which atherosclerosis can be induced adds to its usefulness as a model of human coronary artery d i ~ e a s e The . ~ ~ results ~ of this study have shown their added utility in studies that require the administration of warfarin sodium.
References 1. National Institutes of Health. Guide for the Care and Use of Laboratory Animals. NIH
Publication 85-23; 1985. 2. Physicians Desk Reference. Oradell, NJ: Medical Economics; 1989: pp 902-904. 3. Quick AJ. Hemorrhagic diseases and thrombosis. Philadelphia: Lea 8z Febiger; 1966. 4. Davilla JC, Edds GT, Osuna 0. Modification of the effects of aflatoxin BI and warfarin in young pigs given selenium. Am J Vet Res. 1983;44:1877-1883. 5 . Koph GS, Graeme LH, Geha AS: Long-term performance of the St. Jude Medical
Anticoagulation of Minipigs
145
J Invest Surg Downloaded from informahealthcare.com by Nyu Medical Center on 07/23/15 For personal use only.
valve: Low incidence of thromboembolism and hemorrhagic complications with modest doses of warfarin. Circulation. 1987;76(supplIII):III-132-III-136. 6. Hirsh J. Is the dose of warfarin prescribed by American physicians unnecessarily high? Arch Intern Med. 1987;147:769-771. 7. Kim DN, Lee KT, Schmee J , Thomas WA. Quantification of intimal cell masses and atherosclerotic lesions in coronary arteries of control and hyperlipidemic swine. Atherosclerosis. 1984;52:115- 122. 8. Thomas WA, Kim DN, Lee KT, Reiner JM, Schmee J. Population dynamics of arterial cells during atherogenesis. XIII. Mitogenic and cytotoxic effects of a hyperlipidemic diet on cells in advanced lesions in the abdominal aortas of swine fed HL diet for 270-345 days. Exp Mol Pathol. 1983;39:257-270.
